Electron Microscopic Visualization of Telomerase from Euplotes aediculatus Bound to a Model Telomere DNA

Abstract

Binding of the telomerase ribonucleoprotein from the ciliate Euplotes aediculatus to telomeric DNA in vitro has been examined by electron microscopy (EM). Visualization of the structures that formed revealed a globular protein complex that localized to the DNA end containing the E. aediculatus telomere consensus 3'-single-strand T(4)G(4)T(4)G(4)T(4)G(2) overhang. Gel filtration confirmed that purified E. aediculatus telomerase is an active dimer in solution, and comparison of the size of the DNA-associated complex with apoferritin suggests that E. aediculatus telomerase binds to a single telomeric 3'-end as a dimer. Up to 43% of the telomerase-DNA complexes appeared by EM to involve tetramers or larger multimers of telomerase in association with two or more DNA ends. These data provide the first direct evidence that telomerase is a functional dimer and suggest that two telomerase ribonucleoprotein particles cooperate to elongate each Euplotes telomere in vivo.

Structure of active dimeric human telomerase

“…This mechanism could enable sister telomeres to maintain equal lengths after extension. On the other hand, our data showing that the two TERT subunits in a telomerase dimer co-operate in telomerase activity could also have been explained with a mechanism in which telomerase binds and extends a single DNA substrate in a processive manner, as has been suggested from studies using partly purified Euplotes telomerase 41 . The observed flexibility of the interface between the two monomer lobes in the telomerase structure (Supplementary movie S1) could be functionally important by providing a hinge mechanism for the intramolecular translocation of the DNA substrate from one catalytic pocket to the other, as has been reported for other DNA/RNA polymerases such as the eukaryotic primosome 42 .…”

New Models of Tetrahymena Telomerase RNA from Experimentally Derived Constraints and Modeling

“…4 In fact, no structural data of an intact, minimally functional telomerase complex have been reported except for low-resolution electron microscopic analysis of telomerase isolated from Euplotes aediculatus. 5 Telomerase elongates the linear chromosomes of most eukaryotes with repeating sequences of guanosine-rich DNA to solve the end replication problem faced during DNA replication. 6 Telomerase is critical for the genomic integrity of dividing cells because of its central role in maintaining the chromosome ends.…”

“…Telomerase is an important RNP for which high-resolution structural data of the RNA within the RNP remain incomplete . In fact, no structural data of an intact, minimally functional telomerase complex have been reported except for low-resolution electron microscopic analysis of telomerase isolated from Euplotes aediculatus . Telomerase elongates the linear chromosomes of most eukaryotes with repeating sequences of guanosine-rich DNA to solve the end replication problem faced during DNA replication .…”

Ribonucleoprotein multimers and their functions

“…Gel filtration chromatography of ciliate telomerases from Euplotes crassus and Euplotes aediculatus yielded a molecular weight that is consistent with a dimeric RNP architecture (Aigner et al, 2003; Fouche et al, 2006; Greene and Shippen, 1998; Wang et al, 2002). Intriguingly, Euplotes crassus telomerase can be found in differently sized complexes with distinct biochemical characteristics depending on the developmental stage of the organism, ranging from 280–400kDa in vegetative cells up to >5 MDa in cells undergoing macronuclear development (Greene and Shippen, 1998).…”